Methods for measuring carcinoembryonic antigen

ABSTRACT

Disclosed herein are methods of accurately measuring carcinoembryonic antigen in a biological sample and methods of identifying and treating a mucinous cyst in a subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/092,036 filed Nov. 27, 2013, which claims the benefit of U.S.Provisional Application No. 61/731,725 entitled “Methods for measuringcarcinoembryonic antigen” filed Nov. 30, 2012, U.S. ProvisionalApplication No. 61/824,623 entitled “Methods for measuringcarcinoembryonic antigen” filed May 17, 2013, and U.S. ProvisionalApplication No. 61/840,963 entitled “Methods for measuringcarcinoembryonic antigen” filed Jun. 28, 2013, each of which are herebyincorporated herein by reference in their entirety.

GOVERNMENT INTERESTS

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PARTIES TO A JOINT RESEARCH AGREEMENT

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INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ON A COMPACT DISC

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BACKGROUND

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BRIEF SUMMARY OF THE INVENTION

Embodiments herein are directed to methods of accurately measuringcarcinoembryonic antigen in a biological sample, the method comprising:diluting the biological sample with a diluent to form a diluted samplewhen an amylase level in the biological sample is less than apre-determined amylase threshold between about 10,000 and about 20,000units; wherein diluting the biological sample with a diluent comprisesdiluting the biological sample in a ratio of biological sample todiluent between about 1:10 and about 1:50; and measuringcarcinoembryonic antigen in the diluted sample.

Some embodiments further comprise measuring amylase levels in abiological sample. In some embodiments, the biological sample isundiluted. In some embodiments, the diluent is saline, DiluentUniversal, or a combination thereof. In some embodiments, the biologicalsample is selected from pancreatic fluid, pancreatic cyst fluid andcombinations thereof. In some embodiments, the pre-determined amylasethreshold is about 10,000 units, about 15,000 units, or about 20,000units.

In some embodiments, diluting the biological sample with a diluentcomprises diluting the biological sample with a diluent in a ratio ofbiological sample to diluent of about 1:40. In some embodiments,diluting the biological sample with a diluent comprises diluting thebiological sample with diluent in a ratio of biological sample todiluent of about 1:10.

In some embodiments, the biological sample is not present in sufficientvolume to accurately measure carcinoembryonic antigen levels. In someembodiments, the biological sample has a high viscosity. In someembodiments, the high viscosity is due to the presence of mucin in thebiological sample. In some embodiments, the carcinoembryonic antigenlevels in the biological sample exceed the measurement capabilities ofan instrument being used to measure carcinoembryonic antigen levels.

In some embodiments, the presence of carcinoembryonic antigen above apredetermined carcinoembryonic antigen threshold in the diluted sampleis indicative of a pathological condition. In some embodiments, thecarcinoembryonic antigen level threshold is about 192 ng/ml, or about400 ng/ml. In some embodiments, the pathological condition is selectedfrom a neoplastic condition, a non-neoplastic condition and acombination thereof. In some embodiments, the neoplastic condition isselected from colorectal carcinoma, gastric carcinoma, pancreaticcarcinoma, cholangiocarcinoma, intraductal papillary mucinous neoplasm(IPMN), lung carcinoma, breast carcinoma, medullary thyroid carcinoma,mucinous cystic neoplasm, and combinations thereof. In some embodiments,the non-neoplastic condition is selected from ulcerative colitis,pancreatitis, pancreatic cysts, pancreatic masses, biliary strictures,cirrhosis, chronic obstructive pulmonary disease, Crohn's disease,intraductal papillary mucinous neoplasm (IPMN), and combinationsthereof.

Some embodiments are directed to methods of accurately measuringcarcinoembryonic antigen in a biological sample, the method comprising:diluting the biological sample with a diluent at a ratio of biologicalsample to diluent of about 1:10 to about 1:50 to form a dilutedbiological sample, wherein the amylase level in the biological sample isgreater than a pre-determined amylase threshold between about 10,000 andabout 20,000 units; measuring carcinoembryonic antigen level in thediluted biological sample; multiplying the carcinoembryonic antigenlevel by a correction factor to obtain a corrected carcinoembryonicantigen level.

Some embodiments further comprise measuring amylase levels in abiological sample. In some embodiments, the diluent is saline, DiluentUniversal, or a combination thereof. In some embodiments, the biologicalsample is undiluted. In some embodiments, the biological sample isselected from pancreatic fluid, pancreatic cyst fluid and combinationsthereof.

In some embodiments, the pre-determined amylase threshold is about10,000 units, about 15,000 units, or about 20,000 units. In someembodiments, diluting the biological sample with a diluent comprisesdiluting the biological sample in a ratio of biological sample to salineof about 1:10. In some embodiments, diluting the biological sample witha diluent comprises diluting the biological sample in a ratio ofbiological sample to diluent of about 1:40. In some embodiments, thecorrection factor is about 0.7.

In some embodiments, the biological sample is not present in sufficientvolume to accurately measure carcinoembryonic antigen level. In someembodiments, the biological sample has a high viscosity. In someembodiments, the high viscosity is due to the presence of mucin in thebiological sample. In some embodiments, the carcinoembryonic antigenlevels in the biological sample exceed the measurement capabilities ofan instrument being used to measure carcinoembryonic antigen level.

In some embodiments, a corrected carcinoembryonic antigen level above apredetermined carcinoembryonic antigen threshold is indicative of apathological condition. In some embodiments, the carcinoembryonicantigen level threshold is about 192 ng/ml, or about 400 ng/ml. In someembodiments, the pathological condition is selected from a neoplasticcondition, a non-neoplastic condition and a combination thereof. In someembodiments, the neoplastic condition is selected from colorectalcarcinoma, gastric carcinoma, cholangiocarcinoma, pancreatic carcinoma,intraductal papillary mucinous neoplasm (IPMN), lung carcinoma, breastcarcinoma, medullary thyroid carcinoma, mucinous cystic neoplasm, andcombinations thereof. In some embodiments, the non-neoplastic conditionis selected from ulcerative colitis, pancreatitis, pancreatic cysts,pancreatic masses, biliary strictures, cirrhosis, chronic obstructivepulmonary disease, Crohn's disease, intraductal papillary mucinousneoplasm (IPMN), and combinations thereof.

Some embodiments are directed to methods of identifying a mucinous cystin a subject, the method comprising: diluting the biological sample witha diluent when an amylase level in the biological sample is less than apre-determined amylase threshold between about 10,000 and about 20,000units, to form a diluted sample, wherein diluting the biological samplewith a diluent comprises diluting the biological sample in a ratio ofbiological sample to diluent between about 1:10 and about 1:50; andmeasuring carcinoembryonic antigen level in the diluted sample; whereina carcinoembryonic antigen level above a predetermined carcinoembryonicantigen level threshold in the diluted sample is indicative of amucinous cyst.

Some embodiments further comprise obtaining a biological sample from thesubject. In some embodiments, the diluent is saline, Diluent Universal,or a combination thereof. In some embodiments, the biological sample isselected from pancreatic fluid, pancreatic cyst fluid and combinationsthereof. Some embodiments further comprise measuring amylase levels in abiological sample. In some embodiments, the biological sample isundiluted.

In some embodiments, the pre-determined amylase threshold is about10,000 units, about 15,000 units, or about 20,000 units. In someembodiments, diluting the biological sample with a diluent comprisesdiluting the biological sample in a ratio of biological sample todiluent of about 1:40. In some embodiments, diluting the biologicalsample with a diluent comprises diluting the biological sample in aratio of biological sample to diluent of about 1:10.

In some embodiments, the biological sample is not present in sufficientvolume to accurately measure carcinoembryonic antigen levels. In someembodiments, the biological sample has a high viscosity. In someembodiments, the high viscosity is due to the presence of mucin in theneat biological sample. In some embodiments, the carcinoembryonicantigen levels in the biological sample exceed the measurementcapabilities of an instrument being used to measure carcinoembryonicantigen.

In some embodiments, the carcinoembryonic antigen level threshold isabout 192 ng/ml, or about 400 ng/ml.

Some embodiments are directed to methods of identifying a mucinous cystin a subject, the method comprising: diluting the biological sample witha diluent at a ratio of biological sample to diluent of about 1:10 toabout 1:50 to form a diluted biological sample, wherein the amylaselevels in the biological sample are greater than a pre-determinedamylase threshold between about 10,000 and about 20,000 units; measuringcarcinoembryonic antigen level in the diluted biological sample;multiplying the carcinoembryonic antigen level of the diluted biologicalsample by a correction factor to obtain a corrected carcinoembryonicantigen level, wherein the carcinoembryonic antigen level in the dilutedbiological sample is less than the carcinoembryonic antigen levels in analiquot of the same biological sample diluted with diluent in a ratio ofbiological sample to diluent of about 1:10; and wherein a correctedcarcinoembryonic antigen level of above a predetermined carcinoembryonicantigen threshold is indicative of a mucinous cyst.

Some embodiments further comprise obtaining a biological sample from thesubject. In some embodiments, the diluent is saline, Diluent Universal,or a combination thereof. Some embodiments further comprise measuringamylase levels in a biological sample. In some embodiments, thebiological sample is undiluted. In some embodiments, the biologicalsample is selected from pancreatic fluid, pancreatic cyst fluid andcombinations thereof.

In some embodiments, the pre-determined amylase threshold is about10,000 units, about 15,000 units, or about 20,000 units. In someembodiments, diluting the biological sample with a diluent comprisesdiluting the biological sample in a ratio of biological sample todiluent of about 1:10. In some embodiments, diluting the biologicalsample with diluent comprises diluting the biological sample in a ratioof biological sample to diluent of about 1:40. In some embodiments, thecorrection factor is about 0.7. In some embodiments, thecarcinoembryonic antigen level threshold is about 192 ng/ml or about 400ng/ml.

In some embodiments, the biological sample is not present in sufficientvolume to accurately measure carcinoembryonic antigen. In someembodiments, the biological sample has a high viscosity. In someembodiments, the high viscosity is due to the presence of mucin in theneat biological sample. In some embodiments, the carcinoembryonicantigen levels in the biological sample exceed the measurementcapabilities of an instrument being used to measure carcinoembryonicantigen.

DESCRIPTION OF DRAWINGS

FIG. 1 shows neat carcinoembryonic antigen (CEA) levels in pancreaticcyst fluid samples plotted against CEA levels in pancreatic cyst fluidsamples diluted with saline at a ratio of sample to saline of 1:10.Samples are plotted based on amylase levels (e.g. less than 10,000,15,000, 20,000, 25,000, 30,000 or 40,000)

FIG. 2 shows neat carcinoembryonic antigen (CEA) levels in pancreaticcyst fluid samples plotted against CEA levels in pancreatic cyst fluidsamples diluted with saline at a ratio of sample to saline of 1:10 orwith Diluent Universal at a ratio of sample to diluent of 1:10. Samplesare plotted based on amylase levels (e.g. greater than 15,000, 20,000,25,000, 30,000 or 40,000).

FIG. 3 shows neat carcinoembryonic antigen (CEA) levels in pancreaticcyst fluid samples plotted against CEA levels in pancreatic cyst fluidsamples diluted with saline at a ratio of sample to saline of 1:10 orwith Diluent Universal at a ratio of sample to diluent of 1:10 forsamples with amylase levels greater than 55,000, 60,000, 65,000, 70,000,75,000 or 80,000. Samples are plotted based on amylase levels.

FIG. 4 shows the effect of different correction factors after 1:10dilution with saline of pancreatic cyst fluid with amylase valuesgreater than 15,000 on the ability (accuracy, sensitivity, andspecificity) to detect mucinous pancreatic cysts with CEA values ofgreater than or equal to 192, which can be indicative of the presence ofa mucinous cyst.

FIG. 5 shows the correlation of carcinoembryonic antigen (CEA) levels in266 undiluted cyst fluid samples versus CEA levels in cyst fluid samplesdiluted 1:10 with Diluent Universal (top graph) and 1:10 with DiluentUniversal followed by application of a correction factor of 0.7 forsamples with amylase levels of greater than 15,000 (bottom graph). Theintraclass correlation coefficient (ICC) using Diluent Universal=0.32(0.63 without outlier data point) and intraclass correlation coefficient(ICC) using Diluent Universal followed by application of a correctionfactor of 0.7=0.33 (0.80 without outlier data point). Black crossesrepresent samples with amylase levels equal to, or below 15,000 and graycrosses represent samples with amylase levels greater than 15,000.

FIG. 6 shows the correlation of carcinoembryonic antigen (CEA) levels in266 undiluted cyst fluid samples versus CEA levels in cyst fluid samplesdiluted 1:10 with saline (top graph) and 1:10 with saline followed byapplication of a correction factor of 0.7 for samples with amylaselevels of greater than 15,000 (bottom graph). The intraclass correlationcoefficient (ICC) using saline=0.73 and intraclass correlationcoefficient (ICC) using saline followed by application of a correctionfactor of 0.7=0.84. Black crosses represent samples with amylase levelsequal to or below 15,000 and gray crosses represent samples with amylaselevels greater than 15,000.

FIG. 7 shows Bland-Altman bias plots examining a ratio of undilutedsample CEA levels to diluted sample CEA levels (diluted 1:10 withDiluent Universal) showing statistically significant bias (top graph;bias with outlier data point=0.6553449, p-value<2.2e-16; bias withoutoutlier data point=0.6605572, p-value<2.2e-16), and examined using aratio of undiluted sample CEA levels to diluted sample CEA levels(diluted 1:10 with Diluent Universal followed by application of acorrection factor of 0.7 for samples with amylase levels of greater than15,000) showing a statistically significant but reduced bias (bottomgraph; bias with outlier data point=0.7864442, p-value<2.2e-16; biaswithout outlier data point=0.7932449, p-value<2.2e-16). Black crossesrepresent samples with amylase levels equal to or below 15,000 and graycrosses represent samples with amylase levels greater than 15,000.

FIG. 8 shows Bland Altman bias plots examining a ratio of undilutedsample CEA levels to diluted sample CEA levels (diluted 1:10 withsaline) showing statistically significant bias (top graph;bias=0.8304916, p-value=6.115e-11), and examined using a ratio ofundiluted sample CEA levels to diluted sample CEA levels (diluted 1:10with saline followed by application of a correction factor of 0.7 forsamples with amylase levels of greater than 15,000) showing nostatistically significant bias (bottom graph; bias=0.9966283,p-value=0.8881). Black crosses represent samples with amylase levelsequal to or below 15,000 and gray crosses represent samples with amylaselevels greater than 15,000.

DETAILED DESCRIPTION

Imaging techniques, cytology, and biochemical analysis includingcarcinoembryonic antigen (CEA) concentrations of pancreatic cyst fluidobtained by endoscopic ultrasound-guided fine needle aspiration are usedto differentiate pancreatic cyst lesions, particularly those withmalignant potential. The American College of Gastroenterology guidelineshave outlined the published data relating to the diagnostic performanceof pancreatic cyst fluid CEA and concluded that cyst fluid CEA is thesingle most important factor in determining pancreatic cyst etiology.

In practice, more than 60% of pancreatic cyst and duct fluid CEA testingrequires preliminary sample dilution due to i) low aspirated fluidvolume, ii) high sample viscosity, and/or iii) initial elevated CEAbeyond instrument measurable range (IMR). There are concerns over thefact that CEA measurements in cyst fluid diluted with aqueous saltsolutions or water do not accurately reflect the original, undilutedcyst fluid measurement. Disclosed herein are novel methods that providea more accurate measurement of CEA in diluted pancreatic cyst fluid.

Methods of Accurately Measuring Carcinoembryonic Antigen

Embodiments herein are directed to methods of accurately measuringcarcinoembryonic antigen in a biological sample, the method comprising:diluting the biological sample with a diluent to form a diluted samplewhen an amylase level in the biological sample is less than apre-determined amylase threshold between about 10,000 and about 20,000units; wherein diluting the biological sample with a diluent comprisesdiluting the biological sample in a ratio of biological sample todiluent between about 1:10 and about 1:50; and measuringcarcinoembryonic antigen in the diluted sample. Some embodiments furthercomprise measuring amylase level in a biological sample. In someembodiments, the biological sample is undiluted. In some embodiments,the biological sample is selected from pancreatic fluid, pancreatic cystfluid and combinations thereof. In some embodiments, the pre-determinedamylase threshold is about 10,000 units, about 15,000 units, or about20,000 units. In some embodiments, diluting the biological sample with adiluent comprises diluting the biological sample with diluent in a ratioof biological sample to diluent of about 1:40. In some embodiments,diluting the biological sample with a diluent comprises diluting thebiological sample with a diluent in a ratio of biological sample todiluent of about 1:10. In some embodiments, the diluent is an aqueoussalt solution. Examples of suitable aqueous salt solutions include butare not limited to saline, Diluent Universal (Elecsys Diluent Universal,Roche Diagnostics), Tyrode's solution, lactated Ringer's Solution,acetated Ringer's solution, TRIS-buffered saline (TBS), Hank's balancedsalt solution (HBSS), Earle's balanced salt solution (EBSS), Standardsaline citrate (SSC), HEPES-buffered saline (HBS), Gey's balanced saltsolution (GBSS), minimum essential medium Eagle alpha modification(α-MEM), phosphate buffered saline (PBS), and combinations thereof. Insome embodiments, the biological sample is diluted in water. In someembodiments, the diluent is saline. In some embodiments, the diluent isDiluent Universal (Elecsys Diluent Universal, Roche Diagnostics).

In some embodiments, the biological sample is not present in sufficientvolume to accurately measure carcinoembryonic antigen levels. In someembodiments, the minimum volume to accurately measure CEA is about 200μL. In some embodiments, the biological sample has a high viscosity. Insome embodiments, the high viscosity is due to the presence of mucin inthe biological sample. In some embodiments, the carcinoembryonic antigenlevels in the biological sample exceed the measurement capabilities ofan instrument being used to measure carcinoembryonic antigen levels. Insome embodiments, measuring CEA comprises measuring CEA with a RocheModular Analytics E170 instrument.

In some embodiments, the biological sample is selected from pancreaticfluid, pancreatic cyst fluid and combinations thereof. In someembodiments, the presence of carcinoembryonic antigen above apredetermined carcinoembryonic antigen threshold in the diluted sampleis indicative of a pathological condition. In some embodiments, thecarcinoembryonic antigen level threshold is between about 5 ng/ml andabout 1,000 ng/ml. In some embodiments, the carcinoembryonic antigenlevel threshold is about 192 ng/ml, or about 400 ng/ml. In yet otherembodiments, the carcinoembryonic antigen level threshold is determinedbased on clinical relevance, by the treating physician or a combinationthereof.

In some embodiments, the pathological condition is selected from aneoplastic condition, a non-neoplastic condition and a combinationthereof. In some embodiments, the neoplastic condition is selected fromcolorectal carcinoma, gastric carcinoma, pancreatic carcinoma,cholangiocarcinoma, intraductal papillary mucinous neoplasm (IPMN), lungcarcinoma, breast carcinoma, medullary thyroid carcinoma, mucinouscystic neoplasm, and combinations thereof.

In some embodiments, the non-neoplastic condition is selected fromulcerative colitis, pancreatitis, pancreatic cysts, pancreatic masses,biliary strictures, cirrhosis, chronic obstructive pulmonary disease,Crohn's disease, intraductal papillary mucinous neoplasm (IPMN), andcombinations thereof.

FIG. 1 depicts neat carcinoembryonic antigen (CEA) levels in pancreaticcyst fluid samples plotted against CEA levels in pancreatic cyst fluidsamples diluted with saline at a ratio of sample to saline of 1:10 at avariety of amylase levels (less than 40,000, 30,000, 25,000, 25,000,20,000, 15,000 or 10,000). FIG. 2 depicts neat carcinoembryonic antigen(CEA) levels in pancreatic cyst fluid samples plotted against CEA levelsin pancreatic cyst fluid samples diluted with saline at a ratio ofsample to saline of 1:10 or with Diluent Universal at a ratio of sampleto diluent of 1:10 for samples with amylase levels above 15,000, 20,000,25,000, 30,000, or 40,000. FIG. 3 depicts neat carcinoembryonic antigen(CEA) levels in pancreatic cyst fluid samples plotted against CEA levelsin pancreatic cyst fluid samples diluted with saline at a ratio ofsample to saline of 1:10 or with Diluent Universal at a ratio of sampleto diluent of 1:10 for samples with amylase levels above 55,000, 60,000,65,000, 70,000, 75,000, or 80,000. Applying a correction factor ofaround 0.7 to CEA measurements of cyst fluid specimens with amylasevalues greater than 15000 proved most accurate and sensitive fordetection of mucinous cysts when a mucinous cyst is deemed present basedon CEA levels greater than or equal to 192 ng/mL. In some embodiments,when amylase levels are low (less than 15,000 units), samples dilutedwith a diluent such as but not limited to saline or Diluent Universal ata ratio of sample to diluent of 1:10 results in CEA level measurementsthat are comparable to those in neat pancreatic cyst fluid samples. Ascan be seen in FIGS. 1, 2, and 3, CEA levels in pancreatic cyst fluidsamples diluted with saline at a ratio of sample to saline of 1:10 withamylase levels below 20,000 display a high degree of accuracy to neatcarcinoembryonic antigen (CEA) levels in pancreatic cyst fluid samples.In contrast, CEA levels in pancreatic cyst fluid samples diluted withsaline at a ratio of sample to saline of 1:10 with amylase levels above20,000 display a lesser degree of accuracy to neat carcinoembryonicantigen (CEA) levels in pancreatic cyst fluid samples. The higher theamylase level in a sample, the less accurate the CEA level reading whenthe sample is diluted with saline at a ratio of sample to saline of 1:10compared with neat carcinoembryonic antigen (CEA) levels in pancreaticcyst fluid samples. When amylase levels are high (greater than 15,000units), CEA levels in samples diluted with a diluent such as but notlimited to saline or Diluent Universal at a ratio of sample to saline of1:10 appear to overestimate CEA values compared with undiluted samples.A linear regression analysis of CEA levels from undiluted, neat samplesversus CEA levels in samples diluted in saline at a sample to salineratio of 1:10 reveals that a correction factor of about 0.7 increasesaccuracy of CEA level measurements in samples with amylase levelsgreater than 15,000 units. In addition, FIG. 4 shows that a correctionfactor of 0.7 results in the best accuracy and specificity. In someembodiments, the CEA level obtained in the sample diluted with saline ina sample to saline ratio of 1:10 is multiplied by the correction factor.In some embodiments the correction factor is about 0.7.

In some embodiments, the methods disclosed herein result in an improvedintraclass correlation coefficient. FIG. 5 shows the correlation ofcarcinoembryonic antigen (CEA) levels in 266 undiluted samples versusCEA levels in samples diluted 1:10 with Diluent Universal (top graph)and 1:10 with Diluent Universal followed by application of a correctionfactor of 0.7 for samples with amylase levels of greater than 15,000(bottom graph). The intraclass correlation coefficient (ICC) usingDiluent Universal was 0.32 (0.63 without outlier data point) andintraclass correlation coefficient (ICC) using saline followedapplication of a correction factor of 0.7 was 0.33 (0.80 without outlierdata point).

Bland-Altman bias plots were created examining a ratio of undilutedsample CEA levels to diluted sample CEA levels. A bias score of 1indicated no bias. As can be seen in FIG. 7, the mean bias for cystfluid samples diluted 1:10 with Diluent Universal was 0.6553449(p-value<2.2e-16) (Without outlier data point, bias was 0.6605572,p-value<2.2e-16) indicative of a statistically significant bias (topgraph). In comparison, the mean bias for cyst fluid samples diluted 1:10with Diluent Universal followed by application of a correction factor of0.7 when amylase levels are greater than 15,000 was 0.7864442(p-value<2.2e-16) (without outlier, bias was 0.7932449(p-value<2.2e-16)) indicative of a statistically significant but reducedbias (bottom graph).

FIG. 6 shows the correlation of carcinoembryonic antigen (CEA) levels in266 undiluted samples versus CEA levels in samples diluted 1:10 withsaline (top graph) and 1:10 with saline followed application of acorrection factor of 0.7 for samples with amylase levels of greater than15,000 (bottom graph). The intraclass correlation coefficient (ICC)using saline was 0.73 and the intraclass correlation coefficient (ICC)using saline followed application of a correction factor of 0.7 was0.84.

Bland-Altman bias plots were created examining a ratio of undilutedsample CEA levels to diluted sample CEA levels. A bias score of 1indicated no bias. As can be seen in FIG. 8, the mean bias for cystfluid samples diluted 1:10 with saline was 0.8304916 (p-value=6.15e-11). In comparison, there was no statistically significant bias forcyst fluid samples diluted 1:10 with saline followed by application of acorrection factor of 0.7 when amylase levels are greater than 15,000(0.9966283, p-value=0.8881). These data are indicative that dilution ofsamples with diluents such as, but not limited to saline and DiluentUniversal, followed by application of a correction factor when amylaselevels in the sample are greater than 15,000 results in betterreproduction of CEA measurements in undiluted samples. These data alsoindicate that there is decreased variability (increased ICCmeasurements) and less bias with dilution of samples with diluents suchas, but not limited to saline and Diluent Universal followed byapplication of a correction factor when amylase levels in the sample aregreater than 15,000.

In some embodiments, a correction factor is applied when amylase levelsin a sample diluted 1:40 with diluent are greater than 15,000. In someembodiments, the correction factor is about 0.7. In some embodiments,the diluent is saline, Diluent Universal or a combination thereof. Insome embodiments, a correction factor is applied when amylase levels ina sample diluted 1:10 with diluent are greater than 15,000. In someembodiments, the correction factor is about 0.7. In some embodiments,the diluent is saline, Diluent Universal or a combination thereof. Insome embodiments, a correction factor is applied when amylase levels ina sample diluted 1:40 with saline are greater than 15,000. In someembodiments, the correction factor is about 0.7. In some embodiments, acorrection factor is applied when amylase levels in a sample diluted1:10 with saline are greater than 15,000. In some embodiments, thecorrection factor is about 0.7. Additional studies performed show thecorrelation of carcinoembryonic antigen (CEA) levels in 72 undilutedsamples versus CEA levels in samples diluted 1:10 with saline followedapplication of a correction factor of 0.7 where amylase levels indiluted samples are greater than 15,000. In this study, results were asfollows: ICC=0.91; mean bias=0.94 (p-value=0.139). Yet another studyshows the correlation of carcinoembryonic antigen (CEA) levels in 204undiluted samples versus CEA levels in samples diluted 1:10 with salinefollowed application of a correction factor of 0.7 where amylase levelsin diluted samples are greater than 15,000. In this study, results wereas follows: ICC=0.88; mean bias=1.04 (p-value=0.0996) indicatingimproved variability and no bias when 1:10 dilution is used followed byapplication of a correction factor of 0.7 in samples with amylase levelsabove 15,000. Table 1 illustrates the results from the studies describedherein for various methods of analyzing CEA levels and results indicatethat using an amylase cut off of 15,000 and a sample to saline dilutionof 1:10 to 1:40 wherein a correction factor of 0.7 is applied to sampleswith amylase levels above 15,000 resulted in the superior results andmore accurate CEA measurements compared to not implementing thecorrection factor based on the 15,000 amylase cut off.

TABLE 1 Summary of ICC and Bias for each method of dilution compared toneat CEA values based on not applying (rows 1 and 2) or applying (rows 3and 4) the correction factor of 0.7 based on Amylase cut off of 15,000for specimens diluted 1:10 or 1:40 with saline or diluent universal(UD). Training Set Verification Set w/ out outlier Row # ICC Biasp-value ICC Bias p-value 1 Neat vs. 1:10 Saline 0.87 0.78 9.24E−06 0.790.87 1.54E−05 2 Neat vs. 1:10 UD Not Not Not Done Not Not Done Not DoneDone Done Done 3 Amylase cut-off 0.89 0.98 7.37E−01 0.84 1.13 1.52E−04(1:40) & CF 0.7 4 Amylase cut-off 0.91 0.94 1.39E−01 0.88 1.04 9.96E−02(1:10) & CF 0.7

In some embodiments, where amylase levels in a particular sample arebelow 15,000, the sample is diluted with a diluent at a sample todiluent ratio of 1:40 but no correction factor is used. In someembodiments, where amylase levels in a particular sample are below15,000, the sample is diluted with a diluent at a sample to diluentratio of 1:10 but no correction factor is used.

Some embodiments are directed to methods of accurately measuringcarcinoembryonic antigen in a biological sample, the method comprising:diluting the biological sample with a diluent at a ratio of biologicalsample to diluent of about 1:10 to about 1:50 to form a dilutedbiological sample, wherein the amylase level in the biological sample isgreater than a pre-determined amylase threshold between about 10,000 andabout 20,000 units; measuring carcinoembryonic antigen level in thediluted biological sample; multiplying the carcinoembryonic antigenlevel by a correction factor to obtain a corrected carcinoembryonicantigen level. In some embodiments, the correction factor is about 0.7.

In some embodiments, the correction factor applied may vary. In someembodiments, the correction factor to be applied may range from about0.1 to about 0.8. In some embodiments, the correction factor to beapplied when CEA levels greater than 15,000 is about 0.7. In someembodiments the correction factor to be applied is determined asfollows: a separate linear regression without intercept may be performedby regressing undiluted CEA (neat) values versus CEA values for samplesdiluted in diluent at a sample to diluent ratio of about 1:10 forsubsets of samples where amylase levels is greater than about 15,000.The resulting estimated coefficient from the above-described linearregressions can be used as the correction factor measurement of CEAlevels in a sample diluted with diluent at a sample to diluent ratio of1:10. In some embodiments, the diluent is an aqueous salt solution.Examples of suitable aqueous salt solutions include but are not limitedto saline, Diluent Universal (Elecsys Diluent Universal, RocheDiagnostics), Tyrode's solution, lactated Ringer's Solution, acetatedRinger's solution, TRIS-buffered saline (TBS), Hank's balanced saltsolution (HBSS), Earle's balanced salt solution (EBSS), Standard salinecitrate (SSC), HEPES-buffered saline (HBS), Gey's balanced salt solution(GBSS), minimum essential medium Eagle alpha modification (α-MEM),phosphate buffered saline (PBS), and combinations thereof. In someembodiments, the biological sample is diluted in water. In someembodiments, the diluent is saline. In some embodiments, the diluent isDiluent Universal (Elecsys Diluent Universal, Roche Diagnostics).

In some embodiments the correction factor to be applied is determined asfollows: a separate linear regression without intercept may be performedby regressing undiluted CEA (neat) values versus CEA values for samplesdiluted in a diluent at a sample to diluent ratio of about 1:40 forsubsets of samples where amylase levels is greater than about 15,000.The resulting estimated coefficient from the above-described linearregressions can be used as the correction factor measurement of CEAlevels in a sample diluted with diluent at a sample to diluent ratio of1:40. In some embodiments, the diluent is an aqueous salt solution.Examples of suitable aqueous salt solutions include but are not limitedto saline, Diluent Universal (Elecsys Diluent Universal, RocheDiagnostics), Tyrode's solution, lactated Ringer's Solution, acetatedRinger's solution, TRIS-buffered saline (TBS), Hank's balanced saltsolution (HBSS), Earle's balanced salt solution (EBSS), Standard salinecitrate (SSC), HEPES-buffered saline (HBS), Gey's balanced salt solution(GBSS), minimum essential medium Eagle alpha modification (α-MEM),phosphate buffered saline (PBS), and combinations thereof. In someembodiments, the biological sample is diluted in water. In someembodiments, the diluent is saline. In some embodiments, the diluent isDiluent Universal (Elecsys Diluent Universal, Roche Diagnostics).

Some embodiments further comprise measuring amylase levels in abiological sample. In some embodiments, the biological sample isundiluted. In some embodiments, the biological sample is selected frompancreatic fluid, pancreatic cyst fluid and combinations thereof.

In some embodiments, the pre-determined amylase threshold is about10,000 units, about 15,000 units, or about 20,000 units. In someembodiments, diluting the biological sample with a diluent comprisesdiluting the biological sample in a ratio of sample to diluent of about1:10. In some embodiments, diluting the biological sample with a diluentcomprises diluting the biological sample in a ratio of sample to diluentof about 1:40. In some embodiments, the diluent is an aqueous saltsolution. Examples of suitable aqueous salt solutions include but arenot limited to saline, Diluent Universal (Elecsys Diluent Universal,Roche Diagnostics), Tyrode's solution, lactated Ringer's Solution,acetated Ringer's solution, TRIS-buffered saline (TBS), Hank's balancedsalt solution (HBSS), Earle's balanced salt solution (EBSS), Standardsaline citrate (SSC), HEPES-buffered saline (HBS), Gey's balanced saltsolution (GBSS), minimum essential medium Eagle alpha modification(α-MEM), phosphate buffered saline (PBS), and combinations thereof. Insome embodiments, the biological sample is diluted in water. In someembodiments, the diluent is saline. In some embodiments, the diluent isDiluent Universal (Elecsys Diluent Universal, Roche Diagnostics).

In some embodiments, the biological sample is not present in sufficientvolume to accurately measure carcinoembryonic antigen levels. In someembodiments, the minimum volume to accurately measure CEA is about 200μL. In some embodiments, the biological sample has a high viscosity. Insome embodiments, the high viscosity is due to the presence of mucin inthe biological sample. In some embodiments, the carcinoembryonic antigenlevels in the biological sample exceed the measurement capabilities ofan instrument being used to measure carcinoembryonic antigen levels. Insome embodiments, measuring CEA comprises measuring CEA with a RocheModular Analytics E170 instrument.

In some embodiments, a corrected carcinoembryonic antigen level above apredetermined carcinoembryonic antigen threshold is indicative of apathological condition. In some embodiments, the carcinoembryonicantigen level threshold is between about 5 ng/ml and about 1,000 ng/ml.In some embodiments, the carcinoembryonic antigen level threshold isabout 192 ng/ml, or about 400 ng/ml. In some embodiments, thepathological condition is selected from a neoplastic condition, anon-neoplastic condition and a combination thereof. In some embodiments,the neoplastic condition is selected from colorectal carcinoma, gastriccarcinoma, cholangiocarcinoma, pancreatic carcinoma, intraductalpapillary mucinous neoplasm (IPMN), lung carcinoma, breast carcinoma,medullary thyroid carcinoma, mucinous cystic neoplasm, and combinationsthereof. In some embodiments, the non-neoplastic condition is selectedfrom ulcerative colitis, pancreatitis, pancreatic cysts, pancreaticmasses, biliary strictures, cirrhosis, chronic obstructive pulmonarydisease, Crohn's disease, intraductal papillary mucinous neoplasm(IPMN), and combinations thereof.

Methods of Identifying a Mucinous Cyst

Some embodiments are directed to methods of identifying a mucinous cystin a subject, the method comprising: diluting the biological sample witha diluent when an amylase level in the biological sample is less than apre-determined amylase threshold between about 10,000 and about 20,000units, to form a diluted sample, wherein diluting the biological samplewith a diluent comprises diluting the biological sample in a ratio ofbiological sample to diluent between about 1:10 and about 1:50; andmeasuring carcinoembryonic antigen level in the diluted sample; whereina carcinoembryonic antigen level above a predetermined carcinoembryonicantigen level threshold in the saline diluted sample is indicative of amucinous cyst.

Some embodiments further comprise obtaining a biological sample from thesubject. In some embodiments, the biological sample is selected frompancreatic fluid, pancreatic cyst fluid and combinations thereof. Someembodiments further comprise measuring amylase levels in a biologicalsample. In some embodiments, the biological sample is undiluted.

In some embodiments, the pre-determined amylase threshold is about10,000 units, about 15,000 units, or about 20,000 units. In someembodiments, diluting the biological sample with diluent comprisesdiluting the biological sample in a ratio of sample to diluent of about1:40. In some embodiments, diluting the biological sample with a diluentcomprises diluting the biological sample in a ratio of sample to diluentof about 1:10. In some embodiments, the diluent is an aqueous saltsolution. Examples of suitable aqueous salt solutions include but arenot limited to saline, Diluent Universal (Elecsys Diluent Universal,Roche Diagnostics), Tyrode's solution, lactated Ringer's Solution,acetated Ringer's solution, TRIS-buffered saline (TBS), Hank's balancedsalt solution (HBSS), Earle's balanced salt solution (EBSS), Standardsaline citrate (SSC), HEPES-buffered saline (HBS), Gey's balanced saltsolution (GBSS), minimum essential medium Eagle alpha modification(α-MEM), phosphate buffered saline (PBS), and combinations thereof. Insome embodiments, the biological sample is diluted in water. In someembodiments, the diluent is saline. In some embodiments, the diluent isDiluent Universal (Elecsys Diluent Universal, Roche Diagnostics).

In some embodiments, the carcinoembryonic antigen level threshold isbetween about 5 ng/ml and about 1,000 ng/ml. In some embodiments, thecarcinoembryonic antigen level threshold is about 192 ng/ml, or about400 ng/ml.

In some embodiments, the biological sample is not present in sufficientvolume to accurately measure carcinoembryonic antigen levels. In someembodiments, the biological sample has a high viscosity. In someembodiments, the high viscosity is due to the presence of mucin in theneat biological sample. In some embodiments, the carcinoembryonicantigen levels in the biological sample exceed the measurementcapabilities of an instrument being used to measure carcinoembryonicantigen.

Some embodiments are directed to methods of identifying a mucinous cystin a subject, the method comprising: diluting the biological sample witha diluent at a ratio of biological sample to diluent of about 1:10 toabout 1:50 to form a diluted biological sample, wherein the amylaselevels in the biological sample are greater than a pre-determinedamylase threshold between about 10,000 and about 20,000 units; measuringcarcinoembryonic antigen level in the diluted biological sample;multiplying the carcinoembryonic antigen level of the diluted biologicalsample by a correction factor to obtain a corrected carcinoembryonicantigen level; and wherein a corrected carcinoembryonic antigen level ofabove a predetermined carcinoembryonic antigen threshold is indicativeof a mucinous cyst. In some embodiments, the correction factor is about0.7.

Some embodiments further comprise obtaining a biological sample from thesubject. Some embodiments further comprise measuring amylase levels in abiological sample. In some embodiments, the biological sample isundiluted. In some embodiments, the biological sample is selected frompancreatic fluid, pancreatic cyst fluid and combinations thereof.

In some embodiments, the pre-determined amylase threshold is about10,000 units, about 15,000 units, or about 20,000 units. In someembodiments, diluting the biological sample with a diluent comprisesdiluting the biological sample in a ratio of biological sample todiluent of about 1:10. In some embodiments, diluting the biologicalsample with diluent comprises diluting the biological sample in a ratioof sample to diluent of about 1:40. In some embodiments, the biologicalsample can be diluted in an aqueous salt solution. In some embodiments,the diluent is an aqueous salt solution. Examples of suitable aqueoussalt solutions include but are not limited to saline, Diluent Universal(Elecsys Diluent Universal, Roche Diagnostics), Tyrode's solution,lactated Ringer's Solution, acetated Ringer's solution, TRIS-bufferedsaline (TBS), Hank's balanced salt solution (HBSS), Earle's balancedsalt solution (EBSS), Standard saline citrate (SSC), HEPES-bufferedsaline (HBS), Gey's balanced salt solution (GBSS), minimum essentialmedium Eagle alpha modification (α-MEM), phosphate buffered saline(PBS), and combinations thereof. In some embodiments, the biologicalsample is diluted in water. In some embodiments, the diluent is saline.In some embodiments, the diluent is Diluent Universal (Elecsys DiluentUniversal, Roche Diagnostics).

In some embodiments, the carcinoembryonic antigen level threshold isbetween about 5 ng/ml and about 1,000 ng/ml. In some embodiments, thecarcinoembryonic antigen level threshold is about 192 ng/ml or about 400ng/ml.

In some embodiments, the biological sample is not present in sufficientvolume to accurately measure carcinoembryonic antigen levels. In someembodiments, the minimum volume to accurately measure CEA is about 200μL. In some embodiments, the biological sample has a high viscosity. Insome embodiments, the high viscosity is due to the presence of mucin inthe biological sample. In some embodiments, the carcinoembryonic antigenlevels in the biological sample exceed the measurement capabilities ofan instrument being used to measure carcinoembryonic antigen levels. Insome embodiments, measuring CEA comprises measuring CEA with a RocheModular Analytics E170 instrument.

Methods of Treating a Pathological Condition

Some embodiments are directed to a method of treating a pathologicalcondition in a subject in need thereof, the method comprising measuringCEA in a biological sample from the subject, and administering to thesubject an effective amount of a treatment modality for the pathologywherein the presence of CEA in the biological sample is above apre-determined threshold.

In some embodiments, measuring CEA in a biological sample from thesubject comprises diluting the biological sample with a diluent to forma diluted sample when an amylase level in the biological sample is lessthan a pre-determined amylase threshold between about 10,000 and about20,000 units; wherein diluting the biological sample with diluentcomprises diluting the biological sample in a ratio of biological sampleto diluent between about 1:10 and about 1:50; and measuringcarcinoembryonic antigen in the diluted sample. Some embodiments furthercomprise measuring amylase level in a biological sample. In someembodiments, the pre-determined amylase threshold is about 10,000 units,about 15,000 units, or about 20,000 units.

In some embodiments, the biological sample is undiluted. In someembodiments, the biological sample is selected from pancreatic fluid,pancreatic cyst fluid and combinations thereof. In some embodiments, thebiological sample is selected from pancreatic fluid, pancreatic cystfluid and combinations thereof.

In some embodiments, diluting the biological sample with salinecomprises diluting the biological sample with a diluent in a ratio ofbiological sample to diluent of about 1:40. In some embodiments,diluting the biological sample with a diluent comprises diluting thebiological sample with diluent in a ratio of biological sample todiluent of about 1:10. In some embodiments, the diluent is an aqueoussalt solution. Examples of suitable aqueous salt solutions include butare not limited to saline, Diluent Universal (Elecsys Diluent Universal,Roche Diagnostics), Tyrode's solution, lactated Ringer's Solution,acetated Ringer's solution, TRIS-buffered saline (TBS), Hank's balancedsalt solution (HBSS), Earle's balanced salt solution (EBSS), Standardsaline citrate (SSC), HEPES-buffered saline (HBS), Gey's balanced saltsolution (GBSS), minimum essential medium Eagle alpha modification(α-MEM), phosphate buffered saline (PBS), and combinations thereof. Insome embodiments, the biological sample is diluted in water. In someembodiments, the diluent is saline. In some embodiments, the diluent isDiluent Universal (Elecsys Diluent Universal, Roche Diagnostics).

In some embodiments, the biological sample is not present in sufficientvolume to accurately measure carcinoembryonic antigen levels. In someembodiments, the minimum volume to accurately measure CEA is about 200μL. In some embodiments, the biological sample has a high viscosity. Insome embodiments, the high viscosity is due to the presence of mucin inthe biological sample. In some embodiments, the carcinoembryonic antigenlevels in the biological sample exceed the measurement capabilities ofan instrument being used to measure carcinoembryonic antigen levels. Insome embodiments, measuring CEA comprises measuring CEA with a RocheModular Analytics E170 instrument.

In some embodiments, the presence of carcinoembryonic antigen above apredetermined carcinoembryonic antigen threshold in the saline dilutedsample is indicative of a pathological condition. In some embodiments,the carcinoembryonic antigen level threshold is between about 5 ng/mland about 1,000 ng/ml. In some embodiments, the carcinoembryonic antigenlevel threshold is about 192 ng/ml, or about 400 ng/ml. In someembodiments, the pathological condition is selected from a neoplasticcondition, a non-neoplastic condition and a combination thereof. In someembodiments, the neoplastic condition is selected from colorectalcarcinoma, gastric carcinoma, pancreatic carcinoma, cholangiocarcinoma,intraductal papillary mucinous neoplasm (IPMN), lung carcinoma, breastcarcinoma, medullary thyroid carcinoma, and combinations thereof. Insome embodiments, the non-neoplastic condition is selected fromulcerative colitis, pancreatitis, pancreatic cysts, pancreatic masses,biliary strictures, cirrhosis, chronic obstructive pulmonary disease,Crohn's disease, intraductal papillary mucinous neoplasm (IPMN), andcombinations thereof.

In some embodiments, measuring CEA in a biological sample from thesubject comprises diluting the biological sample with a diluent at aratio of biological sample to diluent of about 1:10 to about 1:50 toform a diluted biological sample, wherein the amylase level in thebiological sample is greater than a pre-determined amylase thresholdbetween about 10,000 and about 20,000 units; measuring carcinoembryonicantigen level in the diluted biological sample; multiplying thecarcinoembryonic antigen level by a correction factor to obtain acorrected carcinoembryonic antigen level. In some embodiments, thecorrection factor is about 0.7.

Some embodiments further comprise measuring amylase levels in abiological sample. In some embodiments, the biological sample isundiluted. In some embodiments, the biological sample is selected frompancreatic fluid, pancreatic cyst fluid and combinations thereof.

In some embodiments, the pre-determined amylase threshold is about10,000 units, about 15,000 units, or about 20,000 units. In someembodiments, diluting the biological sample with diluent comprisesdiluting the biological sample in a ratio of sample to diluent of about1:10. In some embodiments, diluting the biological sample with diluentcomprises diluting the biological sample in a ratio of sample to diluentof about 1:40. In some embodiments, the diluent is an aqueous saltsolution. Examples of suitable aqueous salt solutions include but arenot limited to saline, Diluent Universal (Elecsys Diluent Universal,Roche Diagnostics), Tyrode's solution, lactated Ringer's Solution,acetated Ringer's solution, TRIS-buffered saline (TBS), Hank's balancedsalt solution (HBSS), Earle's balanced salt solution (EBSS), Standardsaline citrate (SSC), HEPES-buffered saline (HBS), Gey's balanced saltsolution (GBSS), minimum essential medium Eagle alpha modification(α-MEM), phosphate buffered saline (PBS), and combinations thereof. Insome embodiments, the biological sample is diluted in water. In someembodiments, the diluent is saline. In some embodiments, the diluent isDiluent Universal (Elecsys Diluent Universal, Roche Diagnostics).

In some embodiments, the biological sample is not present in sufficientvolume to accurately measure carcinoembryonic antigen levels. In someembodiments, the minimum volume to accurately measure CEA is about 200μL. In some embodiments, the biological sample has a high viscosity. Insome embodiments, the high viscosity is due to the presence of mucin inthe biological sample. In some embodiments, the carcinoembryonic antigenlevels in the biological sample exceed the measurement capabilities ofan instrument being used to measure carcinoembryonic antigen levels. Insome embodiments, measuring CEA comprises measuring CEA with a RocheModular Analytics E170 instrument.

In some embodiments, a corrected carcinoembryonic antigen level above apredetermined carcinoembryonic antigen threshold is indicative of apathological condition. In some embodiments, the carcinoembryonicantigen level threshold is between about 5 ng/ml and about 1,000 ng/ml.In some embodiments, the carcinoembryonic antigen level threshold isabout 192 ng/ml, or about 400 ng/ml. In some embodiments, thepathological condition is selected from a neoplastic condition, anon-neoplastic condition and a combination thereof. In some embodiments,the neoplastic condition is selected from colorectal carcinoma, gastriccarcinoma, cholangiocarcinoma, pancreatic carcinoma, intraductalpapillary mucinous neoplasm (IPMN), lung carcinoma, breast carcinoma,medullary thyroid carcinoma, mucinous cystic neoplasm, and combinationsthereof. In some embodiments, the non-neoplastic condition is selectedfrom ulcerative colitis, pancreatitis, pancreatic cysts, pancreaticmasses, biliary strictures, cirrhosis, chronic obstructive pulmonarydisease, Crohn's disease, intraductal papillary mucinous neoplasm(IPMN), and combinations thereof.

In some embodiments, the treatment modality comprises surgicalresection, chemotherapy, drug therapy, biological therapy, gene therapy,vaccine therapy, radiation therapy, photodynamic therapy, hypothermictherapy, laser therapy and combinations thereof.

Some embodiments are directed to a method of treating pancreaticcarcinoma in a subject in need thereof, the method comprising measuringCEA in a biological sample from the subject, and administering to thesubject an effective amount of a treatment modality for the pathologywherein the presence of CEA in the biological sample is above apre-determined threshold.

In some embodiments, measuring CEA in a biological sample from thesubject comprises diluting the biological sample with a diluent to forma diluted sample when an amylase level in the biological sample is lessthan a pre-determined amylase threshold between about 10,000 and about20,000 units; wherein diluting the biological sample with a diluentcomprises diluting the biological sample in a ratio of biological sampleto diluent between about 1:10 and about 1:50; and measuringcarcinoembryonic antigen in the diluted sample.

Some embodiments further comprise measuring amylase level in abiological sample. In some embodiments, the biological sample isundiluted. In some embodiments, the biological sample is selected frompancreatic fluid, pancreatic cyst fluid and combinations thereof.

In some embodiments, the pre-determined amylase threshold is about10,000 units, about 15,000 units, or about 20,000 units. In someembodiments, diluting the biological sample with diluent comprisesdiluting the biological sample with saline in a ratio of biologicalsample with diluent comprises diluting the biological sample withdiluent in a ratio of biological sample to diluent of about 1:40. Insome embodiments, diluting the biological sample with diluent comprisesdiluting the biological sample with diluent in a ratio of biologicalsample to diluent of about 1:10. In some embodiments, the diluent is anaqueous salt solution. Examples of suitable aqueous salt solutionsinclude but are not limited to saline, Diluent Universal (ElecsysDiluent Universal, Roche Diagnostics), Tyrode's solution, lactatedRinger's Solution, acetated Ringer's solution, TRIS-buffered saline(TBS), Hank's balanced salt solution (HBSS), Earle's balanced saltsolution (EBSS), Standard saline citrate (SSC), HEPES-buffered saline(HBS), Gey's balanced salt solution (GBSS), minimum essential mediumEagle alpha modification (α-MEM), phosphate buffered saline (PBS), andcombinations thereof. In some embodiments, the biological sample isdiluted in water. In some embodiments, the diluent is saline. In someembodiments, the diluent is Diluent Universal (Elecsys DiluentUniversal, Roche Diagnostics).

In some embodiments, the biological sample is not present in sufficientvolume to accurately measure carcinoembryonic antigen levels. In someembodiments, the minimum volume to accurately measure CEA is about 200μL. In some embodiments, the biological sample has a high viscosity. Insome embodiments, the high viscosity is due to the presence of mucin inthe biological sample. In some embodiments, the carcinoembryonic antigenlevels in the biological sample exceed the measurement capabilities ofan instrument being used to measure carcinoembryonic antigen levels. Insome embodiments, measuring CEA comprises measuring CEA with a RocheModular Analytics E170 instrument.

In some embodiments, the biological sample is selected from pancreaticfluid, pancreatic cyst fluid and combinations thereof. In someembodiments, the presence of carcinoembryonic antigen above apredetermined carcinoembryonic antigen threshold in the saline dilutedsample is indicative of pancreatic carcinoma. In some embodiments, thecarcinoembryonic antigen level threshold is between about 5 ng/ml andabout 1,000 ng/ml. In some embodiments, the carcinoembryonic antigenlevel threshold is about 192 ng/ml, or about 400 ng/ml.

In some embodiments, measuring CEA in a biological sample from thesubject comprises diluting the biological sample with a diluent at aratio of biological sample to diluent of about 1:10 to about 1:50 toform a diluted biological sample, wherein the amylase level in thebiological sample is greater than a pre-determined amylase thresholdbetween about 10,000 and about 20,000 units; measuring carcinoembryonicantigen level in the diluted biological sample; multiplying thecarcinoembryonic antigen level by a correction factor to obtain acorrected carcinoembryonic antigen level. In some embodiments, thecorrection factor is about 0.7.

Some embodiments further comprise measuring amylase levels in abiological sample. In some embodiments, the biological sample isundiluted. In some embodiments, the biological sample is selected frompancreatic fluid, pancreatic cyst fluid and combinations thereof.

In some embodiments, the pre-determined amylase threshold is about10,000 units, about 15,000 units, or about 20,000 units. In someembodiments, diluting the biological sample with a diluent comprisesdiluting the biological sample in a ratio of sample to diluent of about1:10. In some embodiments, diluting the biological sample with salinecomprises diluting the biological sample in a ratio of sample to diluentof about 1:40. In some embodiments, the diluent is an aqueous saltsolution. Examples of suitable aqueous salt solutions include but arenot limited to saline, Diluent Universal (Elecsys Diluent Universal,Roche Diagnostics), Tyrode's solution, lactated Ringer's Solution,acetated Ringer's solution, TRIS-buffered saline (TBS), Hank's balancedsalt solution (HBSS), Earle's balanced salt solution (EBSS), Standardsaline citrate (SSC), HEPES-buffered saline (HBS), Gey's balanced saltsolution (GBSS), minimum essential medium Eagle alpha modification(α-MEM), phosphate buffered saline (PBS), and combinations thereof. Insome embodiments, the biological sample is diluted in water. In someembodiments, the diluent is saline. In some embodiments, the diluent isDiluent Universal (Elecsys Diluent Universal, Roche Diagnostics).

In some embodiments, the biological sample is not present in sufficientvolume to accurately measure carcinoembryonic antigen levels. In someembodiments, the minimum volume to accurately measure CEA is about 200μL. In some embodiments, the biological sample has a high viscosity. Insome embodiments, the high viscosity is due to the presence of mucin inthe biological sample. In some embodiments, the carcinoembryonic antigenlevels in the biological sample exceed the measurement capabilities ofan instrument being used to measure carcinoembryonic antigen levels. Insome embodiments, measuring CEA comprises measuring CEA with a RocheModular Analytics E170 instrument.

In some embodiments, a corrected carcinoembryonic antigen level above apredetermined carcinoembryonic antigen threshold is indicative ofpancreatic carcinoma. In some embodiments, the carcinoembryonic antigenlevel threshold is between about 5 ng/ml and about 1,000 ng/ml. In someembodiments, the carcinoembryonic antigen level threshold is about 192ng/ml, or about 400 ng/ml.

In some embodiments, the treatment modality comprises surgicalresection, chemotherapy, drug therapy, biological therapy, gene therapy,vaccine therapy, radiation therapy, photodynamic therapy, hypothermictherapy, laser therapy and combinations thereof.

This invention and embodiments illustrating the method and materialsused may be further understood by reference to the followingnon-limiting examples.

EXAMPLES Example 1—Analytical Validation of Dilution of Pancreatic FluidSpecimens for CEA Testing

Purpose

To determine if the New Method for dilution of pancreatic cyst fluidspecimens for CEA testing is improved compared to the clinical methodthrough establishing analytical linearity, accuracy, precision,sensitivity and specificity.

Introduction

CEA measurements in diluted pancreatic cyst fluids have been found tonot accurately reflect the original neat pancreatic cyst fluid CEAvalue. RedPath Integrated Pathology has developed a unique testingmethod to address this issue. A pancreatic cyst fluid is inherently alower volume specimen type, and often times the volume of the specimencollected does not meet the volume requirement for CEA testing. Theability to perform upfront dilutions on pancreatic cysts specimens andprovide an accurate CEA value, as compared to undiluted CEAmeasurements, was the goal in the development of this methodologyimprovement.

CEA Method Development

The development process for this methodology evolved through trial anderror of different applications and the correlating analyses of eachresult set. Pancreatic cyst fluids with excess volume were used for alldevelopmental testing of this method, and dilutions performed onspecimens were compared to the CEA values obtained from testing thespecimens neat (undiluted).

A number of different off-shelf diluents were tested comparatively.During this testing, it was found that Saline showed improvement as adiluent versus Diluent Universal (UD, Roche), which is currently thediluent used to perform dilutions for clinical CEA testing. However, theuse of saline alone was not enough to improve the accuracy of the CEAvalue for all pancreatic cyst fluid specimens when diluted.

An in-house diluent, “Solution CEA”, was developed and tested in effortto improve the accuracy of all diluted pancreatic cyst fluid specimens.This diluent was found to be effective on some specimens but to overperform for certain pancreatic cyst fluid specimens. When specimens weretested in parallel with saline and Solution CEA dilutions thecomparative data was reviewed and a pattern evolved with theincorporation of the Amylase results into the analysis. It wasdetermined that specimens with an Amylase value of less than 15,000 weremore accurate with the use of saline as a diluent when compared to theneat CEA values. However, specimens with an Amylase value of greaterthan 15,000 were more accurate with the use of Solution CEA as thediluent when compared to the neat CEA values.

As more testing was conducted to verify the method and increase the “n”of specimens tested, it was identified through statistical analysis thatthe effect of the Solution CEA as a diluents could be mathematicallyreplicated through the use of a correction factor (CF). Different CFswere trained and tested on varying dilution points. It was found throughthis testing that it was essential to both train and validate the methodon data sets with pancreatic cyst fluids divided proportionately, sincediffer methods of testing were required for specimens based on theAmylase result (please refer to Validation Methods, below).

New CEA Method for Validation

All pancreatic fluid specimens requiring an upfront dilution will bediluted using saline as the diluent, and will be diluted using a 1:10dilution point. Any specimen that requires a dilution and has an Amylasevalue of greater than 15,000 will have a correction factor (CF) of 0.7applied to the CEA value to determine the final CEA result reported.

Example: Amylase=16,000

CEA value after 1:10 dilution=400 (value after adjusted for dilutionfactor)

Final CEA Result for 1:10 dilution=400 multiplied by 0.7=280

Validation Methods

Linearity, Accuracy, Sensitivity & Specificity

To validate the method for dilution of pancreatic cyst fluid specimens,parallel specimen testing for CEA was completed. Each specimen wasdiluted in UD (old method) and saline (new method) with re-testing ofCEA & Amylase on a neat specimen aliquot for result comparison. Thevalidation specimen set was comprised of an equal number of specimensdivided into 4 categories. These four specimen categories are:

-   -   High CEA (greater than 192) & High Amylase (greater than 15,000)    -   Low CEA (less than 192) & High Amylase (greater than 15,000)    -   High CEA (greater than 192) & Low Amylase (less than 15,000)    -   Low CEA (less than 192) & Low Amylase (less than 15,000)

For this validation, 92 pancreatic cyst fluid specimens were accruedwith 23 specimens in each of the four categories described above. Eachspecimen was accrued and tested as follows:

-   -   1. Daily clinical CEA reaction aliquots and Amylase reaction        aliquots were saved that meet the following:        -   a. A NEAT CEA result of 20-1000        -   b. Reportable Amylase result    -   2. All specimen aliquots were stored at 4 C until parallel        testing was completed.    -   3. For each specimen identified, the following testing was        performed:        -   a. Amylase & CEA (on neat specimen) were re-tested→all            remaining specimen volume was saved to perform dilutions            listed below.        -   b. Prepare a 1:10 dilution for CEA using Diluent Universal            -   i. 20 μL of specimen to 180 μL of UD        -   c. Serial Dilution Series “D” for 1:10 & 1:40 dilutions in            Saline were prepared as follows:            -   i. 1:10 dilution for CEA using Saline                -   (1) 30 μL of specimen to 270 μL of Saline            -   ii. 1:40 dilution using Saline & remaining volume from                1:10 dilution in Saline                -   (1) 50 μL of 1:10 in Saline to 150 μL of Saline            -   NOTE: 1:40 not required for this validation.    -   4. Testing was completed as defined by standard operating        procedures for the neat CEA & Amylase re-tests, as well as the        1:10 dilutions in UD and Saline for CEA testing. All testing was        performed on the same day.    -   5. All specimen aliquots, both NEAT and diluted, were stored at        4° C. after testing was completed.    -   6. Specimens were accrued and tested as received.        Precision

To determine precision, pancreatic cyst fluid specimens were collectedto perform parallel specimen testing for CEA. Prior to the precisionstudy, each specimen was re-tested for CEA & Amylase using a neatspecimen aliquot for result comparison. Parallel testing was performedin triplicate on specimen dilutions in UD (old method) and Saline (newmethod). Each dilution was tested over 3 different run scenarios:

-   -   Run 1=DAY 1: Operator 1    -   Run 2=DAY 1: Operator 2    -   Run 3=DAY 2: Operator 1

For this validation, 16 specimens were accrued with 4 specimens in eachof the four categories described above in the “Linearity, Accuracy,Sensitivity & Specificity” section. Each specimen was accrued and testedas follows:

1. Specimens were collected over a period of time that met thefollowing:

a. A NEAT CEA result of 20-1000

b. Reportable Amylase result

c. At least 650 μL of remaining fluid

2. Specimens collected were aliquoted, labeled, and stored at 4° C.

3. Specimens were re-tested to confirm original results prior toproceeding with Step 4, as follows:

a. Re-tested specimens Neat for CEA

b. Re-tested specimens for Amylase

4. Eight specimens with the following (CEA and Amylase clinical reactionaliquots may have been saved and used to re-test CEA and Amylase and/orfor dilution preparations from NEAT reaction aliquots):

a. Clinical Amylase result=LESS THAN 15,000

b. Clinical CEA result=20-1000

-   -   (1) 4 MED/HIGH specimens (192<CEA<400-1000)    -   (2) 4 LOW specimens (CEA<192)        5. Eight specimens with the following (CEA and Amylase clinical        reaction aliquots may have been saved and used to re-test CEA        and Amylase and/or for dilution preparations from NEAT reaction        aliquots):

a. Clinical Amylase result=GREATER THAN 15,000

b. Clinical CEA result=0-1000 (instrument range)

-   -   (1) 4 MED/HIGH specimens (192<CEA<400-1000)    -   (2) 4 LOW specimens (CEA<192)        6. For each specimen identified, triplicate tests were run for        all dilution points (see step 7, below) and tested over a 2-day        period, as listed below:

a. Run 1=DAY 1: Operator 1

b. Run 2=DAY 1: Operator 2

c. Run 3=DAY 2: Operator 1

7. Dilutions for the identified specimens tested as described in step 6are listed below:

a. 1:10 dilution for CEA using UD

-   -   (1) 60 μL of specimen to 540 μL of UD        -   (i) Prepared 3 aliquots (A, B & C) at 200 ul.        -   (ii) 1 Dilution/3 aliquots→3 results

b. Serial Dilution Series “D” as follows:

-   -   (i) 1:10 dilution for CEA using Saline        -   A. 90 μL of specimen to 810 μL of Saline            -   1. Prepared 3 aliquots (A, B & C) at 200 ul.            -   2. 1 Dilution/3 aliquots→3 results    -   (ii) 1:40 dilution using Saline & remaining volume from 1:10        dilution in Saline        -   A. 150 μL of 1:10 in Saline to 450 μL of Saline            -   1. Prepared 3 aliquots (A, B & C) at 200 μL            -   2. 1 Serial Dilution/3 aliquots→3 results

NOTE: 1:40 not required for this validation

8. Testing was completed as defined by SOPs for the neat re-tests forCEA & Amylase testing, as well as the 1:10 dilutions in UD and Salinefor CEA testing. All testing was performed on the same day.

9. All specimen aliquots, both NEAT and diluted, were stored at 4° C.after testing was completed.

Linearity and Accuracy

Intraclass Correlation (ICC), concordance correlation (CC), and biaswere used to measure the agreement between CEA values obtained on neatpancreatic fluid specimens versus diluted pancreatic fluid specimens(both old and new dilution methods). See FIGS. 5 and 6.

The concordance for the old and new dilution method compared to neat(undiluted) CEA values were determine when data was analyzed inclinically relevant categories. The categories were defined by using 192ng/mL as the cut-off between Low CEA and High CEA. A CEA value of 192ng/mL was selected, because this value has been used to discriminatebetween mucinous and non-mucinous cysts. Values at or above 192 ng/mLindicate mucinous cysts; values below this cut-off indicate non-mucinouscysts. The concordance with neat CEA values was improved using the NewCEA method as compared to the Old CEA method (Table 2).

TABLE 2 Concordance between neat CEA values and diluted CEA values (Oldvs. New Method) when data was analyzed in high (>192 ng/mL) and low(<192 ng/mL) CEA categories. Neat CEA values compared to: % ConcordanceOld method 88.04% New Method 91.30%Sensitivity and Specificity

The sensitivity and specificity for measuring the CEA analyte isdescribed by the manufactures of the Elecsys 2010 CEA analyzer (Roche,REF 11731629, http://www.roche.com.mx/fmfiles/re7143001/ElecsysCEA.pdf).The range in which CEA can be measured by this instrument is 0.200-1000ng/mL, with 0.200 being the lower limit of detection. Any interferingsubstances present in pancreatic cyst fluid will be less abundant whendilution in saline occurs.

Precision

Coefficient of variation (CV) was used to assess the reproducibility ofCEA values obtained on neat pancreatic fluid specimens versus dilutedpancreatic fluid specimens (both old and new dilution methods). Pleasesee Table 3 for the inter-run and intra-run reproducibility of olddilution method and Table 4 for inter-run and intra-run reproducibilityof new dilution method.

TABLE 3 Inter-Run & Intra-Run Reproducibility-Old CEA Dilution MethodOverall Inter Run 4.16% Overall Intra Run 2.85% MEAN CV MEAN CV OverallInter Run 4.00% Overall Intra Run 2.16% Median CV Median CV

TABLE 4 Inter-Run Reproducibility-New CEA Dilution Method Overall InterRun 4.72% Overall Intra Run 2.50% MEAN CV MEAN CV Overall Inter Run4.67% Overall Intra Run 2.03% Median CV Median CVValidation ConclusionsLinearity & Accuracy

There is an increase in agreement between the neat CEA value and thediluted CEA value using the New CEA Dilution method versus the Old CEAdilution method. Intraclass Correlation (ICC), concordance correlation(CC), bias, and categorical concordance between diluted and neat CEAvalues were all improved using the New CEA dilution method as comparedto the Old CEA dilution method.

Sensitivity & Specificity

Sensitivity and Specificity are per the manufacturer's description forthe Elecsys 2010 (Roche) instrument CEA analyzer.

Precision

Both methods showed good reproducibility, with the New CEA Dilutionmethod showing a slight improvement in overall intra-runreproducibility. Inter-run reproducibility is generally accepted withCVs of less than 15%; intra-run reproducibility is generally acceptedwith CVs of less than 10%. All CVs for the New CEA dilution method wereless than these values.

The results described above, indicate that the New CEA Dilution methodperformance is similar to or improved compared to the current clinicalmethod (i.e. Old CEA Dilution method) for pancreatic fluid samples.Therefore the New CEA Dilution method is acceptable for implementationon clinical pancreatic fluid specimens received for CEA testing.

REFERENCES

-   1. Clinical Chemistry 2010; 56; 1351-1352-   2. Gastroenterol 2004; 126: 1330-1336-   3. AM J Gastroenterol 2007; 102:2339-2349-   4.    http://www.salimetrics.com/documents/spit-tips/publications/Inter%20and%20Intra%20Assay%20Coefficients%20of%20Variability.pdf

The present disclosure is not to be limited in terms of the particularembodiments described in this application, which are intended asillustrations of various aspects. Many modifications and variations canbe made without departing from its spirit and scope, as will be apparentto those skilled in the art. Functionally equivalent methods andapparatuses within the scope of the disclosure, in addition to thoseenumerated herein, will be apparent to those skilled in the art from theforegoing descriptions. Such modifications and variations are intendedto fall within the scope of the appended claims. The present disclosureis to be limited only by the terms of the appended claims, along withthe full scope of equivalents to which such claims are entitled. It isto be understood that this disclosure is not limited to particularmethods, reagents, compounds, compositions or biological systems, whichcan, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodimentsonly, and is not intended to be limiting.

With respect to the use of substantially any plural and/or singularterms herein, those having skill in the art can translate from theplural to the singular and/or from the singular to the plural as isappropriate to the context and/or application. The varioussingular/plural permutations may be expressly set forth herein for sakeof clarity.

It will be understood by those within the art that, in general, termsused herein, and especially in the appended claims (e.g., bodies of theappended claims) are generally intended as “open” terms (e.g., the term“including” should be interpreted as “including but not limited to,” theterm “having” should be interpreted as “having at least,” the term“includes” should be interpreted as “includes but is not limited to,”etc.). It will be further understood by those within the art that if aspecific number of an introduced claim recitation is intended, such anintent will be explicitly recited in the claim, and in the absence ofsuch recitation no such intent is present. For example, as an aid tounderstanding, the following appended claims may contain usage of theintroductory phrases “at least one” and “one or more” to introduce claimrecitations. However, the use of such phrases should not be construed toimply that the introduction of a claim recitation by the indefinitearticles “a” or “an” limits any particular claim containing suchintroduced claim recitation to embodiments containing only one suchrecitation, even when the same claim includes the introductory phrases“one or more” or “at least one” and indefinite articles such as “a” or“an” (e.g., “a” and/or “an” should be interpreted to mean “at least one”or “one or more”); the same holds true for the use of definite articlesused to introduce claim recitations. In addition, even if a specificnumber of an introduced claim recitation is explicitly recited, thoseskilled in the art will recognize that such recitation should beinterpreted to mean at least the recited number (e.g., the barerecitation of “two recitations,” without other modifiers, means at leasttwo recitations, or two or more recitations). Furthermore, in thoseinstances where a convention analogous to “at least one of A, B, and C,etc.” is used, in general such a construction is intended in the senseone having skill in the art would understand the convention (e.g., “asystem having at least one of A, B, and C” would include but not belimited to systems that have A alone, B alone, C alone, A and Btogether, A and C together, B and C together, and/or A, B, and Ctogether, etc.). In those instances where a convention analogous to “atleast one of A, B, or C, etc.” is used, in general such a constructionis intended in the sense one having skill in the art would understandthe convention (e.g., “a system having at least one of A, B, or C” wouldinclude but not be limited to systems that have A alone, B alone, Calone, A and B together, A and C together, B and C together, and/or A,B, and C together, etc.). It will be further understood by those withinthe art that virtually any disjunctive word and/or phrase presenting twoor more alternative terms, whether in the description, claims, ordrawings, should be understood to contemplate the possibilities ofincluding one of the terms, either of the terms, or both terms. Forexample, the phrase “A or B” will be understood to include thepossibilities of “A” or “B” or “A and B.”

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

As will be understood by one skilled in the art, for any and allpurposes, such as in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” and the like include the number recited andrefer to ranges, which can be subsequently broken down into subranges asdiscussed above. Finally, as will be understood by one skilled in theart, a range includes each individual member. Thus, for example, a grouphaving 1-3 substituents refers to groups having 1, 2, or 3 substituents.Similarly, a group having 1-5 substituents refers to groups having 1, 2,3, 4, or 5 substituents, and so forth.

What is claimed:
 1. A method of measuring carcinoembryonic antigen in abiological sample, the method comprising: diluting the biological samplewith a diluent at a ratio of biological sample to diluent of about 1:10to about 1:50 to form a diluted biological sample; measuring the amylaselevel of the diluted biological sample; and measuring carcinoembryonicantigen level in the diluted biological sample, wherein the measuredcarcinoembryonic antigen amount is not adjusted with a correctionfactor.
 2. The method of claim 1, wherein the diluent is saline, DiluentUniversal, or a combination thereof.
 3. The method of claim 1, whereinthe biological sample is undiluted prior to the diluting step.
 4. Themethod of claim 1, wherein diluting the biological sample with a diluentcomprises diluting the biological sample with a diluent in a ratio ofabout 1:10.
 5. The method of claim 1, wherein diluting the biologicalsample with a diluent comprises diluting the biological sample with adiluent in a ratio of about 1:40.
 6. The method of claim 1, wherein thebiological sample is not present in sufficient volume to accuratelymeasure carcinoembryonic antigen level prior to the diluting step. 7.The method of claim 1, wherein the biological sample has a highviscosity prior to the diluting step.
 8. The method of claim 7, whereinthe high viscosity is due to the presence of mucin in the biologicalsample.
 9. The method of claim 1, wherein the carcinoembryonic antigenlevels in the biological sample exceed the measurement capabilities ofan instrument being used to measure carcinoembryonic antigen level priorto the diluting step.
 10. The method of claim 1, wherein the biologicalsample is selected from pancreatic fluid, pancreatic cyst fluid andcombinations thereof.